Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

A broad-spectrum macrocyclic peptide inhibitor of the SARS-CoV-2 spike protein.

The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used messenger RNA (mRNA) display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor-binding domain, N-terminal domain, and S2 region, distal to the angiotensin-converting enzyme 2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target.

Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design.

The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.

Ancestral Sequence Reconstruction Identifies Structural Changes Underlying the Evolution of Ideonella sakaiensis PETase and Variants with Improved Stability and Activity.

The improved production, recycling, and removal of plastic waste, such as polyethylene terephthalate (PET), are pressing environmental and economic issues for society. Biocatalytic (enzymatic) PET depolymerization is potentially a sustainable, low-energy solution to PET recycling, especially when compared with current disposal methods such as landfills, incineration, or gasification. IsPETase has been extensively studied for its use in PET depolymerization; however, its evolution from cutinases is not fully understood, and most engineering studies have neglected the majority of the available sequence space remote from the active site. In this study, ancestral protein reconstruction (ASR) has been used to trace the evolutionary trajectory from ancient serine hydrolases to IsPETase, while ASR and the related design approach, protein repair one-stop shop, were used to identify enzyme variants with improved activity and stability. Kinetic and structural characterization of these variants reveals new insights into the evolution of PETase activity and the role of second-shell mutations around the active site. Among the designed and reconstructed variants, we identified several with melting points 20 °C higher than that of IsPETase and two variants with significantly higher catalytic activity.

A Comprehensive Phylogenetic Analysis of the Serpin Superfamily.

Serine protease inhibitors (serpins) are found in all kingdoms of life and play essential roles in multiple physiological processes. Owing to the diversity of the superfamily, phylogenetic analysis is challenging and prokaryotic serpins have been speculated to have been acquired from Metazoa through horizontal gene transfer due to their unexpectedly high homology. Here, we have leveraged a structural alignment of diverse serpins to generate a comprehensive 6,000-sequence phylogeny that encompasses serpins from all kingdoms of life. We show that in addition to a central "hub" of highly conserved serpins, there has been extensive diversification of the superfamily into many novel functional clades. Our analysis indicates that the hub proteins are ancient and are similar because of convergent evolution, rather than the alternative hypothesis of horizontal gene transfer. This work clarifies longstanding questions in the evolution of serpins and provides new directions for research in the field of serpin biology.

Postoperative Use of Ketorolac Improves Pain Management and Decreases Narcotic Use Following Primary Cleft Palate Surgery.

OBJECTIVE: To study the efficacy and safety profile of ketorolac in cleft palate surgery. DESIGN: Retrospective analysis of patients who underwent primary cleft palate surgery and received either postoperative ketorolac or opioids. SETTING: Tertiary care children's hospital. PATIENTS, PARTICIPANTS: Eighty-nine patients enrolled who were all younger than 36 months of age, not dependent on a gastrostomy tube, with no history of bleeding disorders, and had undergone their primary cleft palate procedure by one specific surgeon between January 2010 and June 2019. INTERVENTIONS: n/a. MAIN OUTCOME MEASURE: Morphine equivalent dose (MED), Face, Legs, Activity, Cry, Consolability (FLACC) score, length of stay (LOS), total oral intake (mL), total oral intake/LOS, and postoperative adverse events between ketorolac and no ketorolac groups. RESULTS: MED, FLACC score, and LOS were significantly lower in the ketorolac group compared to the no ketorolac group. One patient in the ketorolac group had a bleeding event. CONCLUSIONS: Use of ketorolac significantly decreased narcotic usage and pain scores as reported by the FLACC score. Moreover, postoperative bleeding was rare in both ketorolac and no ketorolac groups.

micro-RNA-486-5p protects against kidney ischemic injury and modifies the apoptotic transcriptome in proximal tubules.

Acute kidney injury (AKI) carries high morbidity and mortality, and effective treatments are lacking. Preclinical models support involvement of micro-RNAs (miRs) in AKI pathogenesis, although effects on the kidney transcriptome are unclear. We previously showed that injection of cord blood endothelial colony forming cell-derived exosomes, enriched in miR-486-5p, prevented ischemic AKI in mice. To further define this, we studied direct effects of miR-486-5p in mice with kidney ischemia-reperfusion injury. RNA-Seq was used to compare the impact of miR-486-5p and exosomes on the transcriptome of proximal tubules and kidney endothelial cells 24 hours after ischemia-reperfusion. In mice with AKI, injection of miR-486-5p mimic increased its levels in proximal tubules and endothelial cells, and improved plasma creatinine, histological injury, neutrophil infiltration, and apoptosis. Additionally, miR-486-5p inhibited expression of its target phosphatase and tensin homolog, and activated protein kinase B. In proximal tubules, miR-486-5p or exosomes reduced expression of genes associated with ischemic injury and the tumor necrosis factor (TNF) pathway, and altered distinct apoptotic genes. In endothelial cells, genes associated with metabolic processes were altered by miR-486-5p or exosomes, although TNF pathway genes were not affected. Thus, our results suggest that miR-486-5p may have therapeutic potential in AKI.

Sex diversity in proximal tubule and endothelial gene expression in mice with ischemic acute kidney injury.

Female sex protects against development of acute kidney injury (AKI). While sex hormones may be involved in protection, the role of differential gene expression is unknown. We conducted gene profiling in male and female mice with or without kidney ischemia-reperfusion injury (IRI). Mice underwent bilateral renal pedicle clamping (30 min), and tissues were collected 24 h after reperfusion. RNA-sequencing (RNA-Seq) was performed on proximal tubules (PTs) and kidney endothelial cells. Female mice were resistant to ischemic injury compared with males, determined by plasma creatinine and neutrophil gelatinase-associated lipocalin (NGAL), histologic scores, neutrophil infiltration, and extent of apoptosis. Sham mice had sex-specific gene disparities in PT and endothelium, and male mice showed profound gene dysregulation with ischemia-reperfusion compared with females. After ischemia PTs from females exhibited smaller increases compared with males in injury-associated genes lipocalin-2 (Lcn2), hepatitis A virus cellular receptor 1 (Havcr1), and keratin 18 (Krt18), and no up-regulation of SRY-Box transcription factor 9 (Sox9) or keratin 20 (Krt20). Endothelial up-regulation of adhesion molecules and cytokines/chemokines occurred in males, but not females. Up-regulated genes in male ischemic PTs were linked to tumor necrosis factor (TNF) and Toll-like receptor (TLR) pathways, while female ischemic PTs showed up-regulated genes in pathways related to transport. The data highlight sex-specific gene expression differences in male and female PTs and endothelium before and after ischemic injury that may underlie disparities in susceptibility to AKI.

Protein engineering: the potential of remote mutations.

Engineered proteins, especially enzymes, are now commonly used in many industries owing to their catalytic power, specific binding of ligands, and properties as materials and food additives. As the number of potential uses for engineered proteins has increased, the interest in engineering or designing proteins to have greater stability, activity and specificity has increased in turn. With any rational engineering or design pursuit, the success of these endeavours relies on our fundamental understanding of the systems themselves; in the case of proteins, their structure-dynamics-function relationships. Proteins are most commonly rationally engineered by targeting the residues that we understand to be functionally important, such as enzyme active sites or ligand-binding sites. This means that the majority of the protein, i.e. regions remote from the active- or ligand-binding site, is often ignored. However, there is a growing body of literature that reports on, and rationalises, the successful engineering of proteins at remote sites. This minireview will discuss the current state of the art in protein engineering, with a particular focus on engineering regions that are remote from active- or ligand-binding sites. As the use of protein technologies expands, exploiting the potential improvements made possible through modifying remote regions will become vital if we are to realise the full potential of protein engineering and design.

A retrospective chart review assessing antibiotic treatment of hospitalized patients with discordant Clostridioides difficile assays in an urban hospitalized setting.

Clostridioides difficile infection (CDI) threatens vulnerable populations in health care. Two-step testing improves specificity, avoiding over-treatment. This study analyzed inpatient records to estimate diagnostic outcomes and identify characteristics associated with treatment after discordant testing. Among discordant patients, those aged 65+ years were significantly more likely to be prescribed antibiotics (67% vs 39%).

Rugged fitness landscapes minimize promiscuity in the evolution of transcriptional repressors.

How a protein's function influences the shape of its fitness landscape, smooth or rugged, is a fundamental question in evolutionary biochemistry. Smooth landscapes arise when incremental mutational steps lead to a progressive change in function, as commonly seen in enzymes and binding proteins. On the other hand, rugged landscapes are poorly understood because of the inherent unpredictability of how sequence changes affect function. Here, we experimentally characterize the entire sequence phylogeny, comprising 1,158 extant and ancestral sequences, of the DNA-binding domain (DBD) of the LacI/GalR transcriptional repressor family. Our analysis revealed an extremely rugged landscape with rapid switching of specificity, even between adjacent nodes. Further, the ruggedness arises due to the necessity of the repressor to simultaneously evolve specificity for asymmetric operators and disfavors potentially adverse regulatory crosstalk. Our study provides fundamental insight into evolutionary, molecular, and biophysical rules of genetic regulation through the lens of fitness landscapes.

Academic Half Day Improves Resident Perception of Education Without Compromising Patient Safety.

BACKGROUND: Residency programs are required to offer a didactic curriculum and protect resident time for education. Our institution implemented an academic half day (AHD) in the 2021-2022 academic year to address issues related to the standard noon conference series. OBJECTIVE: Determine the impact of AHD implementation on education, patient safety, and workflow. METHODS: This was a prospective, single-site educational intervention study. Pre- and post-implementation surveys and Accreditation Council for Graduate Medical Education (ACGME) surveys assessed changes in trainee and faculty attitudes and behaviors. Patient safety and workflow were evaluated by comparing the number of safety event reports, rapid response team activations, time to admission from the ED, and time of discharge on AHD days compared to other weekdays. RESULTS: Survey response rates were: residents 68%/48%, fellows 42%/35%, and faculty 59%/29%. AHD was associated with a significant, positive change in resident attitudes and experiences and on ACGME survey items. On AHDs compared with other weekdays, there were no significant differences in safety event report rates (P = .98), nor in rapid response team activation rates (P = .99). There was not a clinically meaningful difference in median admission time from the ED on AHD weekdays (125 minutes) compared to other weekdays (130 minutes, P = .04). There was no significant difference in median discharge time on AHD vs other weekdays (P = .13). CONCLUSIONS: This study suggests that there is no significant difference in patient safety or workflow with the implementation of AHD. This study supports prior studies that residents strongly prefer AHD. AHD may be a useful framework for resident education without compromising patient care.

The more you know: Insights from integrated pre-visit surveys in a pediatric environmental health center.

The Pediatric Environmental Health Center (PEHC) at Boston Children's Hospital is a specialty referral clinic that provides consultation for approximately 250 patients annually. Identifying environmental hazards is key for clinical management. Exposure concerns include lead, mold, pesticides, perfluoroalkyl substances (PFAS), impaired air quality, and more. Our goal was to identify concerns and visit priorities of our patient population to guide visits. A 47-question pre-visit survey was created exploring potential environmental hazards and administered prior to visits using a platform integrated into the electronic medical record (EMR). The study group was a convenience sample of patients from June 2021 to June 2022. Of 204 total visits, 101 surveys were submitted, yielding a response rate of 49.5%. 66/101 (65.3%) were surveys from initial consultations used for descriptive analysis. The majority of patients were seen for a chief complaint of lead exposure (90.1%). Most respondents had concerns about peeling paint (40.0%), and those reporting peeling paint were more likely to report additional concerns [75.0%, p < 0.001]. Other concerns highlighted were mold (15.2%), pests (15.2%), asbestos (10.6%), air pollution (9.1%), temperature regulation (7.6%), pesticides (6.1%), PFAS (4.5%), and formaldehyde (4.5%). A knowledge gap was identified; 45.5% (30/66) respondents responded "no" to the question asking if the Poison Control Center phone number was stored in their phone. This study illustrates how the implementation of a pre-visit EMR integrated survey engages families, informs clinical care, and serves as a point-of-care education tool for specific knowledge gaps. Findings will guide development of future environmental health screeners.

Evo-velocity: Protein language modeling accelerates the study of evolution.

Understanding how protein sequences have evolved is one of the defining challenges in modern biology. In this issue of Cell Systems, Hie et al. describe a novel phylogenetic approach, dubbed "evo-velocity," that exploits protein language modeling to overcome many limitations of traditional phylogenetic analysis.

Comprehensive phylogenetic analysis of the ribonucleotide reductase family reveals an ancestral clade.

Ribonucleotide reductases (RNRs) are used by all free-living organisms and many viruses to catalyze an essential step in the de novo biosynthesis of DNA precursors. RNRs are remarkably diverse by primary sequence and cofactor requirement, while sharing a conserved fold and radical-based mechanism for nucleotide reduction. Here, we structurally aligned the diverse RNR family by the conserved catalytic barrel to reconstruct the first large-scale phylogeny consisting of 6779 sequences that unites all extant classes of the RNR family and performed evo-velocity analysis to independently validate our evolutionary model. With a robust phylogeny in-hand, we uncovered a novel, phylogenetically distinct clade that is placed as ancestral to the classes I and II RNRs, which we have termed clade Ø. We employed small-angle X-ray scattering (SAXS), cryogenic-electron microscopy (cryo-EM), and AlphaFold2 to investigate a member of this clade from Synechococcus phage S-CBP4 and report the most minimal RNR architecture to-date. Based on our analyses, we propose an evolutionary model of diversification in the RNR family and delineate how our phylogeny can be used as a roadmap for targeted future study.

Billions of years ago, the Earth's atmosphere had very little oxygen. It was only after some bacteria and early plants evolved to harness energy from sunlight that oxygen began to fill the Earth's environment. Oxygen is highly reactive and can interfere with enzymes and other molecules that are essential to life. Organisms living at this point in history therefore had to adapt to survive in this new oxygen-rich world. An ancient family of enzymes known as ribonucleotide reductases are used by all free-living organisms and many viruses to repair and replicate their DNA. Because of their essential role in managing DNA, these enzymes have been around on Earth for billions of years. Understanding how they evolved could therefore shed light on how nature adapted to increasing oxygen levels and other environmental changes at the molecular level. One approach to study how proteins evolved is to use computational analysis to construct a phylogenetic tree. This reveals how existing members of a family are related to one another based on the chain of molecules (known as amino acids) that make up each protein. Despite having similar structures and all having the same function, ribonucleotide reductases have remarkably diverse sequences of amino acids. This makes it computationally very demanding to build a phylogenetic tree. To overcome this, Burnim, Spence, Xu et al. created a phylogenetic tree using structural information from a part of the enzyme that is relatively similar in many modern-day ribonucleotide reductases. The final result took seven continuous months on a supercomputer to generate, and includes over 6,000 members of the enzyme family. The phylogenetic tree revealed a new distinct group of ribonucleotide reductases that may explain how one adaptation to increasing levels of oxygen emerged in some family members, while another adaptation emerged in others. The approach used in this work also opens up a new way to study how other highly diverse enzymes and other protein families evolved, potentially revealing new insights about our planet's past.

Analysis of insertions and extensions in the functional evolution of the ribonucleotide reductase family.

Ribonucleotide reductases (RNRs) are used by all free-living organisms and many viruses to catalyze an essential step in the de novo biosynthesis of DNA precursors. RNRs are remarkably diverse by primary sequence and cofactor requirement, while sharing a conserved fold and radical-based mechanism for nucleotide reduction. In this work, we expand on our recent phylogenetic inference of the entire RNR family and describe the evolutionarily relatedness of insertions and extensions around the structurally homologous catalytic barrel. Using evo-velocity and sequence similarity network (SSN) analyses, we show that the N-terminal regulatory motif known as the ATP-cone domain was likely inherited from an ancestral RNR. By combining SSN analysis with AlphaFold2 predictions, we also show that the C-terminal extensions of class II RNRs can contain folded domains that share homology with an Fe-S cluster assembly protein. Finally, using sequence analysis and AlphaFold2, we show that the sequence motif of a catalytically essential insertion known as the finger loop is tightly coupled to the catalytic mechanism. Based on these results, we propose an evolutionary model for the diversification of the RNR family.

The effect of nutritional status on post-operative outcomes in pediatric otolaryngology-head and neck surgery.

INTRODUCTION: Nutritional status can affect surgical patients in terms of stress response, healing time, and outcomes. Several abnormalities are known to have a high prevalence in the general population such as vitamin D deficiency (VDD) and subclinical hypothyroidism. We hypothesized that there will be elevated rates of nutritional deficiencies in preoperative patients which may adversely affect postoperative outcomes following pediatric otolaryngology surgery. METHODS: IRB approval was obtained for a cross-sectional cohort study. Consecutive patients underwent nutritional evaluation when being scheduled for surgery including TSH, albumin and vitamin D. Demographic data, supplementation, and early complication rates were collected. RESULTS: 125 patients were included in the final cohort with adequate demographic distribution. Based on anthropometric data, 12% of our cohort was found to be undernourished, and 40% of our cohort with elevated BMI. However, there was no relationship found between Z-scores and complications. VDD was noted in 83/125 (66.4%) patients. Our cohort had increased rates of VDD in patients with elevated BMI and African American ethnicity. Thyroid hormone abnormalities were present in 12 patients. Mean serum albumin level was 4.29 in our cohort all within normal range. We did find increased risk of postoperative complications in patients with previously diagnosed comorbidities. (p=0.006). CONCLUSION: There is no current recommendation or consensus for nutritional assessment in preoperative pediatric patients. Our study did not show statistically significant correlation with z-scores, low vitamin D levels with supplementation, albumin, or TSH to postoperative complications. However, our patient cohort had higher than average rates of VDD compared to the many studies of the general pediatric population and significant negative correlation between vitamin D levels and z-scores. By early preoperative identification of VDD and supplementation with calciferol, we found no significant difference in complication rates in patients based on their initial vitamin D status. We suggest screening preoperative patients using z-score calculations and vitamin D levels based on individual patient risk factors including atrisk patient populations such as African American children, and obese children.

Ancestral sequence reconstruction for protein engineers.

In addition to its value in the study of molecular evolution, ancestral sequence reconstruction (ASR) has emerged as a useful methodology for engineering proteins with enhanced properties. Proteins generated by ASR often exhibit unique or improved activity, stability, and/or promiscuity, all of which are properties that are valued by protein engineers. Comparison between extant proteins and evolutionary intermediates generated by ASR also allows protein engineers to identify substitutions that have contributed to functional innovation or diversification within protein families. As ASR becomes more widely adopted as a protein engineering approach, it is important to understand the applications, limitations, and recent developments of this technique. This review highlights recent exemplifications of ASR, as well as technical aspects of the reconstruction process that are relevant to protein engineering.

Pharmacists' nonprescription syringe dispensing perceptions and behaviors: A three-state descriptive analysis.

BACKGROUND: One approach to increasing the reach of syringe programs in rural areas could be through provision of syringes at community pharmacies. This study evaluated relationships between state-specific syringe policies, pharmacy, and pharmacist characteristics and pharmacists' nonprescription syringe dispensing behaviors in a 3- state Appalachian region at high risk for HIV and HCV transmission. METHODS: We conducted a telephone census of community pharmacies in the Appalachian counties of North Carolina, Tennessee, and Virginia from April-June 2018. Behaviors studied included having ever sold syringes without a prescription, quantity of individuals to whom nonprescription syringes were dispensed in the past 30 days, having ever denied a request for nonprescription syringes, and past 30-day denial of nonprescription syringe requests. Behavioral intention and perceptions of legality were elicited. RESULTS: A response rate of 52.3 % was achieved (N = 391). North Carolina pharmacists reported increased past 30-day dispensing, less denial of nonprescription syringe requests, and decreased justification for syringe dispensing (proof of medical need) as compared to Tennessee and Virginia pharmacists. Behavioral intention to dispense did not vary by state but did vary by political affiliation. Perceptions of syringe dispensing legality in NC were significantly different from those in TN and VA. CONCLUSIONS: Significant differences in pharmacists' perceptions and behaviors were noted across state lines with North Carolina pharmacists reporting more engagement in syringe dispensing as compared to pharmacists in Tennessee and Virginia. Policy allowing pharmacists to dispense syringes to people who inject drugs appears to foster some but not all pharmacist engagement in this harm reduction intervention.